In vitro production of a cell population using feeder cells

Abstract

A method for the in vitro production of a cell population P′ from a cell population P, the production requiring the presence of at least one factor which is expressed by feeder cells, wherein a) feeder cells proliferate at a temperature T 1 , b) proliferated feeder cells are contacted with the cell population P, c) the cell mixture obtained at step (b) is cultivated at a temperature T 2 which is chosen such that the cell population P proliferates and the feeder cells do not proliferate, the at least one factor being expressed by the feeder cells, and d) the cell population P′ so produced is recovered. Advantageously, the production consists in an expansion, the feeder cells are insect feeder cells and the cell population P to be expanded is a T lymphocyte population, preferably a Trl lymphocyte population.

The invention claimed is: 
     
       1. A method for the in vitro expansion of a mammal Tr1 cell population P′, from a purified or enriched mammal Tr1 regulatory cell population P, in a culture medium Mp, said expansion requiring the presence of at least five factors in said culture medium Mp, said method comprising:
 (a) cultivating insect feeder cells at a temperature T 1  and in a culture medium Mf, said insect feeder cells capable of expressing said at least five factors, the T 1  being a temperature that allows proliferation of said feeder cells, and said at least five factors comprise:
 (i) an anti-CD3 monoclonal antibody anchored to the insect feeder cell membrane, and 
 (ii) a CD80 protein, a CD86 protein or an anti-CD28 monoclonal antibody, said CD80 protein, CD86 protein or anti-CD28 monoclonal antibody being anchored to the insect feeder cell membrane and capable of cross-linking a CD28 molecule; 
 (iii) a CD58 protein anchored to the cell membrane of the insect feeder cells, the CD58 protein being capable of interacting with the CD2 protein of the Tr1 cells, 
 (iv) an IL-2 secreted by the insect feeder cells, the IL-2 being capable of interacting with the IL-2 receptor of the Tr1 cells, and 
 (v) an interleukin selected from the group consisting of IL-4 and IL-13, said interleukin being secreted by the insect feeder cells and capable of interacting with the IL-4 receptor of the Tr1 cells; 
 
 (b) contacting the insect feeder cells obtained at step (a) with the Tr1 cell population P in the culture medium Mp, wherein said culture medium Mp does not initially contain the at least five factors, to obtain a mixture containing the Tr1 cell population P, the insect feeder cells and the culture medium Mp, 
 c) cultivating the mixture obtained at step (b), wherein the at least five factors are expressed by the insect feeder cells in the culture medium Mp, and said cultivating is carried out at a temperature T 2 , said temperature T 2  being chosen such that:
 the Tr1 cell population P proliferates, and 
 the insect feeder cells do not proliferate, thus expanding the Tr1 cell population P′; and 
 
 d) recovering the Tr1 cell population P′ so expanded,
 wherein said method is capable of maintaining exponential growth of the Tr1 cell population P′ for at least one month. 
 
 
     
     
       2. The method of  claim 1 , wherein the insect feeder cells are recombinant cells and contain one or more heterologous nucleic acid encoding said at least five factors. 
     
     
       3. The method of  claim 1 , wherein T 1  is less than T 2  and T 2  is at least about 35° C. 
     
     
       4. The method of  claim 1 , wherein the culture medium Mp is a serum-free culture medium and the culture medium Mf is a serum-free culture medium. 
     
     
       5. The method of  claim 1 , wherein the Tr1 cell population P′ is an antigen-specific Tr1 cell population. 
     
     
       6. The method of  claim 1 , wherein the Tr1 cell population P comprises antigen-specific Tr1 cells, and the antigen-specific Tr1 cell population P′ is an antigen-specific expanded Tr1 cell population. 
     
     
       7. The method of  claim 1 , wherein said mammal Tr1 cells are human Tr1 cells and the at least five factors are of human origin. 
     
     
       8. The method of  claim 7 , wherein:
 a light chain of the anti-CD3 antibody anchored to the insect feeder cell membrane is encoded by the heterologous nucleic acid of sequence SEQ ID NO: 1, and 
 a heavy chain of the anti-CD3 antibody anchored to the insect feeder cell membrane is encoded by the heterologous nucleic acid of sequence SEQ ID NO: 2. 
 
     
     
       9. The method of  claim 1 , wherein the CD80 protein is encoded by the nucleic acid comprising sequence SEQ ID NO: 3. 
     
     
       10. The method of  claim 1 , wherein the CD86 protein is encoded by the nucleic acid comprising SEQ ID NO: 4. 
     
     
       11. The method of  claim 1 , wherein the IL-2 is encoded by the nucleic acid comprising SEQ ID NO: 5. 
     
     
       12. The method of  claim 1 , wherein the CD58 protein is encoded by the nucleic acid comprising SEQ ID NO: 6. 
     
     
       13. The method of  claim 1 , wherein the IL-4 is encoded by the nucleic acid comprising SEQ ID NO: 7. 
     
     
       14. The method of  claim 1 , wherein the IL-13 is encoded by the nucleic acid comprising SEQ ID NO: 8. 
     
     
       15. The method of  claim 1 , wherein the Tr1 cell population P′ is recovered at step (d) after having cultivated the Tr1 cell population in the mixture at step (c) for at least 12 hours. 
     
     
       16. The method of  claim 1 , wherein the anti-CD3 monoclonal antibody is a modified anti-CD3 antibody, the modification of the anti-CD3 antibody being a replacement of the anti-CD3 intracytoplasmic domain of the anti-CD3 heavy chain with a transmembrane domain, said modified anti-CD3 antibody being susceptible to interact with a CD3/TCR protein complex of the T cells. 
     
     
       17. The method of  claim 1 , wherein the anti-CD3 monoclonal antibody is a modified anti-CD3 antibody, the modification of the anti-CD3 antibody being a replacement of the anti-CD3 intracytoplasmic domain of the anti-CD3 heavy chain with a transmembrane domain of a platelet derived growth factor (PDGF) receptor. 
     
     
       18. The method of  claim 1 , wherein the insect feeder cells do not have any intrinsic class I and/or class II major histocompatibility complex (MHC) molecule at the cell surface. 
     
     
       19. The method of  claim 1 , wherein the insect feeder cells are from the S2 Drosophila cell line deposited on Mar. 25, 2005, at the National Collection of Micro-organisms Cultures (CNCM) under the number 1-3407. 
     
     
       20. A method for the in vitro expansion of a mammal Tr1 cell population P′, from a purified or enriched mammal Tr1 cell population P, in a culture medium Mp, said expansion requiring the presence of at least five factors in said culture medium Mp, said method comprising:
 a) cultivating insect feeder cells at a temperature T 1  in a culture medium Mf, said insect feeder cells capable of expressing said at least five factors, the T 1  being a temperature that allows proliferation of said feeder cells, and said at least five factors comprise:
 (i) an anti-CD3 monoclonal antibody anchored to the insect feeder cell membrane, 
 (ii) a CD80, a CD86 protein, or an anti-CD28 monoclonal antibody, said CD80 protein, CD86 protein or anti-CD28 monoclonal antibody being anchored to the insect feeder cell membrane and capable of cross-linking a CD28 molecule, 
 (iii) a CD58 protein anchored to the insect feeder cell membrane, the CD58 protein being capable of interacting with the CD2 protein of the Tr1 cells, 
 (iv) an IL-2 secreted by the insect feeder cells, the IL-2 being capable of interacting with the IL-2 receptor of the Tr1 cells, and 
 (v) an interleukin selected from the group consisting of IL-4 and IL-13, said interleukin being secreted by the insect feeder cells and being capable of interacting with the IL-4 receptor of the Tr1 cells; 
 
 b) contacting the insect feeder cells obtained at step (a) with the Tr1 cell population P in the culture medium Mp, wherein said culture medium Mp does not initially contain the at least five factors, in order to obtain a mixture containing the Tr1 cell population P, the insect feeder cells and the culture medium Mp, 
 c) cultivating the mixture obtained at step (b), wherein the at least five factors are expressed by the insect feeder cells in the culture medium Mp, and said cultivating is carried out at a temperature T 2 , said temperature T 2  being chosen such that:
 the Tr1 cell population P proliferates, and 
 the insect feeder cells do not proliferate, thus expanding the Tr1 cell population P′, and 
 
 d) recovering the Tr1 cell population P′ so expanded,
 wherein said method is capable of expanding the Tr1 cell population P′ to obtain at least 10 9  cells. 
 
 
     
     
       21. The method of  claim 20 , wherein the insect feeder cells do not have any intrinsic class I and/or class II major histocompatibility complex (MHC) molecule at the cell surface.